The goal of this project is to identify key factors that regulate the genetically programmed switches in beta-like globin gene activity that occur during human development. Elucidation of these control factors may suggest approaches to therapeutically manipulate the expression of the epsilon or gamma genes in ways that are beneficial to individuals with sickle cell anemia and beta thalassemia. Because the control of globin switching is complex, precise identification of the regulators has proven difficult. This project employs three comparative approaches to pinpoint these regulators. First, phylogenetic footprinting takes advantage of the fact that the beta-like globin clusters of the placental mammals were derived from the same five gene cluster and that similar developmental switches in gene activity occur in all mammalian lineages. Since key components of the switching machinery must thus be conserved, aligned beta cluster sequences from primates and other mammals are used to identify short, anciently conserved cis elements within neutral DNA. The trans factors which bind to these elements are then characterized by gel shift assays. Application of this strategy to the epsilon and gamma globin genes has revealed several potentially important interactions, including multiple binding sites for both a repressor protein and for a putative stage-selector protein. The role of these proteins in the developmental expression of epsilon and gamma will be directly tested in this proposal by in vivo footprinting and by expression studies in transgenic mice. Additional important regions of the globin cluster will also be analyzed by phylogenetic footprinting (LCR, beta promoter beta 3' enhancer). A second approach, differential phylogenetic footprinting, may reveal the cis and trans factors involved in the recruitment of simian (anthropoid) gamma genes to a fetal expression pattern. By comparing sequences from simian (fetal gamma) and non-simian (embryonic gamma) primates, base differences associated with this alteration in gamma expression are identified and functional tests (binding and expression studies) are used to test this association. One set of cis changes has been identified near the CCAAT box of the gamma gene that alters the pattern of trans factor binding (to simian vs. non-simian probes) and changes the relative expression levels of simian vs. non-simian promoters in erythroid cells. To determine whether these cis changes also modify the pattern of developmental expression, they will be tested in transgenic mice. Additionally, the search for anthropoid-specific elements will be expanded to other relevant areas of the globin cluster (LCR, beta promoter). Finally, initial sequence data suggest that fine tuning of the two gamma genes has proceeded along different lines among the anthropoid primates: in platyrrhines, gamma2 is the major fetally expressed gene, while in catarrhines, gamma1 predominates. Protein and DNA sequencing studies will be used to determine gamma1 and gamma2 levels in fetal and embryonic blood of several platyrrhine species. The sequence differences between platyrrhines with high gamma2 and catarrhines may reveal additional elements which modulate gamma gene expression; elucidation of such factors is important to the ultimate goal of manipulation of gamma gene activity in individuals with defective adult globin genes.